A decellularized dermal matrix, its preparation method and application

Abstract

This invention belongs to the field of biomaterials technology, specifically relating to an acellular dermal matrix, its preparation method, and its application. The preparation method includes: placing discarded giant nevus tissue removed from a patient in a cold chain preservation solution; removing the epidermis and fat layer under aseptic conditions to obtain pretreated tissue; subjecting the pretreated giant nevus tissue to decellularization and depigmentation treatments to obtain depigmented giant nevus tissue; and finally, stabilization and sterilization to obtain the acellular dermal matrix. This invention uses autologous giant nevus tissue as a donor, which can completely remove melanin and nevus cells from the tissue, effectively reducing the risk of giant nevus recurrence and melanin deposition; at the same time, it can completely preserve the natural structure and biomechanical properties of the dermal matrix, utilizing waste materials and providing reliable biomechanical support for subsequent reimplantation of the acellular dermal matrix.

Description

Technical Field

[0001] This invention belongs to the field of biomaterials technology, specifically relating to a decellularized dermal matrix, its preparation method, and its application. Background Technology

[0002] Giant congenital melanocytic nevus (GCMN) is a type of congenital melanocytic nevus, typically ≥100 cm² in size. 2 Giant nevi can cover 80% or more of the body surface area, severely affecting patients' appearance and quality of life. In addition to potentially causing symptoms such as ulceration, pain, and itching, the chance of giant nevi transforming into melanoma is as high as 5% to 10%, and about 70% of malignant transformations occur in childhood and adolescence. Therefore, early intervention for giant nevi is of great significance in ensuring the safety of patients' lives.

[0003] Currently, surgical excision is the preferred treatment for giant nevi, but the repair of large postoperative wounds remains a challenge in clinical treatment. Various flaps and skin grafts are the mainstream repair methods: flap repair has good aesthetic results, but the donor area is limited, making it difficult to meet the needs of repairing large-area wounds; skin grafts (especially split-thickness grafts) can be harvested multiple times and the donor site scar is not obvious, so they are often used for repairing large-area wounds, but there are problems such as significant post-transplantation pigmentation, poor elasticity, poor texture, and severe contracture, resulting in unsatisfactory postoperative repair outcomes.

[0004] In recent years, acellular dermal matrix (ADM) has demonstrated excellent biocompatibility and tissue regeneration potential as a skin substitute material in the repair of burns, trauma, and chronic wounds. The dermal matrix structure of ADM can serve as a scaffold for angiogenesis and cell migration, effectively promoting wound repair. Studies have shown that ADM combined with split-thickness skin grafts can improve repair outcomes in several ways: firstly, the intact collagen and matrix structure of ADM can serve as a natural scaffold for cell adhesion and angiogenesis, significantly improving the survival rate of split-thickness skin grafts; secondly, it can reduce fluid accumulation between the skin graft and the wound base, lowering the risk of necrosis and infection; and thirdly, it can inhibit skin graft contraction and reduce scar formation, resulting in softer, more elastic skin after repair, with an appearance closer to normal skin.

[0005] Currently, the widespread clinical use of allogeneic ADM has many drawbacks, mainly including: 1. Limited sources and high costs: It relies on donor tissue, which may involve ethical issues. Moreover, the source is scarce and expensive, making it difficult to promote widely. Patients with large-area wound repair are prone to insufficient donors and a heavy economic burden; 2. Risk of immune rejection: Even after decellularization, allogeneic ADM may still trigger a host immune response, affecting the survival of the skin graft; 3. Risk of disease transmission: Allogeneic tissue may carry pathogens, posing a risk of infectious disease transmission, especially for diseases with unknown pathogenesis; 4. Risk of microbial contamination: The sources of allogeneic ADM are complex, and it is easily contaminated by microorganisms during transportation, storage and other processes, which may lead to local or systemic infections.

[0006] Therefore, there is an urgent need to develop a low-cost, non-immune rejection, non-disease transmission, and low infection risk autologous depigmentation-acellular dermal matrix (MFADM) for giant nevi. Summary of the Invention

[0007] Based on the above technical background, the main objective of this invention is to provide a decellularized dermal matrix, its preparation method, and its application, so as to overcome the shortcomings of the prior art.

[0008] To achieve the aforementioned objectives, the technical solution adopted by this invention includes:

[0009] The first aspect of this invention is to provide a method for preparing decellularized dermal matrix, the preparation process of which is illustrated in the schematic diagram below. Figure 1 As shown, the preparation method includes the following steps:

[0010] Step 1: Place the giant nevus tissue in a cold chain preservation solution and remove the epidermis and fat layer of the giant nevus tissue under sterile conditions to obtain the pretreated giant nevus tissue.

[0011] Step 2: Repeatedly freeze and thaw the pretreated giant nevus tissue, and then treat it with ultrasonic vibration to obtain decellularized giant nevus tissue;

[0012] Step 3: Depigment the decellularized giant nevus tissue to obtain depigmented giant nevus tissue;

[0013] Step 4: Stabilize the depigmented giant nevus tissue and then sterilize it to obtain decellularized dermal matrix.

[0014] The steps described above are described in detail below.

[0015] In step 1, the giant nevus tissue is derived from discarded giant nevus tissue removed by the patient.

[0016] The cold chain preservation solution includes physiological saline and antibiotics, wherein the antibiotics are a penicillin-streptomycin mixed solution.

[0017] The penicillin-streptomycin mixed solution (dual antibody) is a 100× concentrate, i.e., 10,000 U / mL penicillin + 10 mg / mL streptomycin. When using, dilute 1× to obtain a final concentration of 100 U / mL penicillin + 100 μg / mL streptomycin.

[0018] The antibiotic has a mass concentration of 0.7-1.5% in the cold chain preservation solution.

[0019] Preferably, the antibiotic has a mass concentration of 1.0% in the cold chain preservation solution.

[0020] The specific procedure for removing giant nevus tissue is as follows: first, all fatty tissue is removed by trimming with ophthalmic scissors, then the epidermis is completely peeled off with a dermal scalpel, and finally a 1 mm thick dermal tissue is cut as experimental material.

[0021] In step 2, the repeated freeze-thaw cycles include sequential freezing and thawing, with the number of freeze-thaw cycles being 3 to 5.

[0022] Preferably, the freeze-thaw cycle is repeated 4 times.

[0023] The conditions for repeated freeze-thaw cycles are: freezing at -90 to -70°C for 5 to 7 hours, followed by thawing at 35 to 40°C for 0.5 to 2 hours (a single freeze-thaw cycle includes one freezing and one thawing).

[0024] Preferably, the conditions for repeated freeze-thaw cycles are: freezing at -80°C for 6 hours, followed by thawing at 37°C for 1 hour, with a total of 4 freeze-thaw cycles.

[0025] The conditions for ultrasonic oscillation are as follows: the giant nevus tissue after repeated freeze-thaw cycles is placed in sterile isotonic saline elution solution for ultrasonic oscillation at a frequency of 30-50 kHz. The sterile isotonic saline elution solution is replaced every 5-7 hours, and the oscillation is performed continuously for 70-75 hours.

[0026] Preferably, the conditions for ultrasonic oscillation are as follows: the giant nevus tissue after repeated freeze-thaw cycles is placed in sterile isotonic saline elution solution for ultrasonic oscillation at a frequency of 40 kHz, the sterile isotonic saline elution solution is changed every 6 h, and ultrasonic oscillation is performed continuously for 72 h.

[0027] This invention employs a physical method involving repeated freeze-thaw cycles and ultrasonic vibration to decellularize giant nevus tissue. The decellularization process used in this invention can preserve the natural bioactivity and structural integrity of the decellularized dermal matrix (MFADM) to the greatest extent possible, while avoiding the side effects caused by chemical reagent residues or biological enzymes.

[0028] In step 3, the decolorization treatment includes decolorization treatment with hydrogen peroxide (H2O2) or decolorization treatment with acidic bleach.

[0029] Preferably, the decolorization treatment is a hydrogen peroxide decolorization treatment.

[0030] Preferably, the conditions for the hydrogen peroxide depigmentation treatment are as follows: using hydrogen peroxide with a mass concentration of 1-3%, treating at a temperature of 35-40°C for 20-60 minutes, followed by multiple washes with isotonic physiological saline.

[0031] More preferably, the conditions for the hydrogen peroxide decolorization treatment are as follows: using hydrogen peroxide with a mass concentration of 2%, treating at a temperature of 37°C for 40 min, followed by multiple washes with isotonic physiological saline.

[0032] Preferably, the conditions for the acidic bleaching depigmentation treatment are as follows: using a chlorine-containing bleach (sodium hypochlorite NaClO) with a mass concentration of 1-3%, treating at a temperature of 35-40°C for 20-60 minutes, followed by multiple washes with isotonic physiological saline.

[0033] More preferably, the conditions for the acidic bleaching depigmentation treatment are as follows: using a 2% (w / w) chlorine-containing bleach (sodium hypochlorite NaClO), treating at 37°C for 40 min, followed by multiple washes with isotonic saline.

[0034] In step 4, the stabilization treatment conditions are as follows: the depigmented giant nevus tissue is uniformly irradiated with ultraviolet light at a wavelength of 365 nm for 20–40 seconds. Uniform irradiation with ultraviolet light allows the giant nevus tissue to form a covalently cross-linked network.

[0035] Preferably, the stabilization treatment conditions are as follows: the depigmented giant nevus tissue is uniformly irradiated with ultraviolet light with a wavelength of 365nm for 30 seconds.

[0036] After stabilization treatment, sterilization is performed using the ethylene oxide method, which includes the following steps:

[0037] (1) Inspection and loading of samples to be sterilized: Confirm that the depigmented and stabilized giant nevus tissue is free of obvious stains and dry; wrap it with breathable packaging material (such as Tyvek paper), leaving gaps during loading to ensure gas flow; at the same time check the airtightness of the sterilizer, the pressure system and the leakage of the ethylene oxide storage tank, and empty the residual gas in the sterilization chamber.

[0038] (2) Pretreatment: Adjust the temperature of the sterilization chamber to 30-60℃ and the relative humidity to 30%-80% to improve the sterilization effect; make the temperature of the items to be sterilized consistent with the temperature of the sterilization chamber environment to avoid the temperature difference affecting the gas permeability.

[0039] (3) Vacuuming: Start the vacuuming program of the sterilizer to reduce the pressure in the sterilization chamber, exhaust the air to reserve space for ethylene oxide gas injection, and at the same time enhance the gas penetration.

[0040] (4) Gas injection and sterilization: According to the volume of the items and sterilization requirements, inject ethylene oxide gas in a quantitative amount (usually 800-1200 mg / L); maintain the set temperature and pressure conditions, sterilize for 1-6 hours to ensure that microorganisms are completely killed.

[0041] (5) Ventilation analysis: After sterilization, first exhaust the ethylene oxide gas in the sterilization room (which can be recovered or treated harmlessly); introduce clean air to replace and ventilate multiple times to analyze the residual gas on the surface and inside of the items until the residual amount is lower than the safety standard (e.g., medical supplies residue ≤10 μg / g).

[0042] The preparation method described in this invention can remove cells and pigments while maintaining the integrity and mechanical strength of decellularized matrix collagen fibers and tissue structure. It can be used for wound repair after surgical excision of giant nevi, and can also avoid recurrence or immune response caused by residual nevus cells or pigments.

[0043] A second aspect of the present invention is to provide a decellularized dermal matrix prepared by the preparation method described in the first aspect of the present invention.

[0044] A third aspect of the present invention is to provide an application of the decellularized dermal matrix described in the second aspect of the present invention in the field of biomaterials.

[0045] Specifically, the decellularized dermal matrix can be used for wound repair after surgical excision of giant nevi.

[0046] The beneficial effects of this invention are as follows:

[0047] (1) This invention utilizes the patient's own excised giant nevus tissue to prepare autologous acellular dermal matrix (MFADM), which can completely avoid the risk of immune rejection of allogeneic materials and has excellent biocompatibility; at the same time, it makes full use of the surgically excised discarded giant nevus tissue as a donor, effectively solving the problem of insufficient skin supply for the repair of large-area wounds. Through the synergistic treatment of decellularization and depigmentation, the integrity of the dermal structure and biomechanical properties can be preserved, while reducing the risk of giant nevus recurrence and melanin deposition.

[0048] (2) The preparation method of this invention mainly uses physical methods to achieve decellularization and depigmentation of giant nevus tissue, which can preserve the integrity of the dermal matrix structure to the greatest extent, reduce the amount of chemical reagents used, avoid the toxicity of chemical reagent residues to the decellularized dermal matrix, and ensure its clinical application effect. The decellularized dermal matrix prepared by this invention has an intact three-dimensional network structure and uniform pore distribution, providing a stable scaffold for cell attachment and migration; histological staining results confirm that the nevus cells in the matrix are completely removed and the collagen fiber structure is well preserved, providing reliable biomechanical support for subsequent transplantation; at the same time, it has excellent biocompatibility, which is conducive to the growth and proliferation of fibroblasts and meets the needs of wound repair.

[0049] (3) The present invention adopts a physical decellularization method of "repeated freeze-thaw + ultrasonic oscillation" combined with hydrogen peroxide or sodium hypochlorite depigmentation treatment, which can avoid chemical reagent residues, retain collagen fibers to the maximum extent, and thoroughly remove melanin and nevus cells in giant nevus tissue, effectively reduce the risk of malignant transformation, and improve the wound repair effect and skin appearance quality.

[0050] (4) The preparation method of this invention is simple and easy to implement, and the equipment used is conventional. It can establish a standardized preparation process for autologous MFADM derived from giant nevi, providing technical assurance for its safety and effectiveness in wound repair. The prepared acellular dermal matrix is ​​expected to achieve the goals of reducing the risk of malignant transformation in patients with giant nevi, improving the level of functional recovery and skin appearance, and improving patients' mental health and quality of life. At the same time, the application of autologous MFADM can make full use of the patient's own waste tissue, get rid of dependence on expensive allogeneic materials, and has significant economic benefits and promotional value. Attached Figure Description

[0051] Figure 1 A schematic flowchart of the method for preparing decellularized dermal matrix according to the present invention is shown;

[0052] Figure 2The images show various stages of the autologous MFADM preparation process for giant nevus tissue: Image A shows a photograph of the giant nevus tissue; Images B-F show photographs after decellularization and depigmentation treatment, with the operation sequence being degreasing (Image B), epidermal removal (Image C), decellularization (Image D), washing (Image E), and depigmentation (Image F); In Images A-F, the left side shows a photograph of depigmentation using H2O2 (Example 1), and the right side shows a photograph of depigmentation using NaClO (Example 2);

[0053] Figure 3 Scanning electron micrographs of untreated giant nevus tissue, and decellularized dermal matrix prepared in Examples 1 and 2 are shown.

[0054] Figure 4 The results of the biocompatibility test of the decellularized dermal matrix prepared in Examples 1 and 2 are shown in Experimental Example 2;

[0055] Figure 5 The results of compression, stretching, and stretching cycle tests are shown for normal skin tissue and decellularized dermal matrix prepared in Examples 1 and 2. Detailed Implementation

[0056] The present invention will now be described in detail, and its features and advantages will become clearer and more apparent from these descriptions.

[0057] Example

[0058] The present invention is further illustrated below with specific examples. These embodiments are merely illustrative and not intended to limit the scope of the invention. All raw materials used in the embodiments of the present invention (except for giant nevus tissue) were commercially available.

[0059] Example 1

[0060] A method for preparing a decellularized dermal matrix, the method comprising the following steps:

[0061] 1. Pretreatment of giant nevus tissue: The giant nevus tissue was placed in a cold chain preservation solution (composed of physiological saline and a penicillin-streptomycin mixture, the stock solution of which was a 100× concentrate, i.e., 10,000 U / mL penicillin + 10 mg / mL streptomycin, diluted 1× before use, with a final concentration of 100 U / mL penicillin + 100 μg / mL streptomycin; the mass concentration of antibiotics in the cold chain preservation solution was 1.0%). Under aseptic conditions, the epidermis and fat layer were removed. Specifically, all fat tissue was removed with ophthalmic scissors, the epidermis was thoroughly peeled off with a dermatome, and finally a 1 mm thick dermis was cut as experimental material.

[0062] 2. Decellularization: The pretreated giant nevus tissue was subjected to repeated freeze-thaw cycles (frozen at -80℃ for 6 h, thawed at 37℃ for 1 h, each freeze-thaw cycle included one freeze-thaw cycle and one thaw cycle, for a total of 4 cycles); then it was placed in sterile isotonic saline elution buffer and subjected to ultrasonic agitation (frequency 40 kHz, elution buffer changed every 6 h, continuous agitation and elution for 72 h) to obtain the decellularized giant nevus tissue.

[0063] 3. Depigmentation treatment: The decellularized giant nevus tissue was treated with 2% hydrogen peroxide solution at 37°C for 40 min, and then washed repeatedly with isotonic saline to obtain the depigmented giant nevus tissue.

[0064] 4. Stabilization and sterilization: The depigmented giant nevus tissue was uniformly irradiated with ultraviolet light at a wavelength of 365 nm for 30 seconds for stabilization treatment; then sterilized by ethylene oxide method to obtain decellularized dermal matrix. Figure 2 The photos on the left side of A to F in this embodiment are photos of each stage of the preparation process.

[0065] Example 2

[0066] The decellularized dermal matrix was prepared in a manner similar to that in Example 1, with the only difference being:

[0067] The depigmentation treatment used a 2% (w / w) chlorine bleaching agent (sodium hypochlorite, NaClO) at 37°C for 40 min, followed by multiple washes with isotonic saline.

[0068] Figure 2 The right-hand photos of A through F in this embodiment are photos of each stage of the preparation process.

[0069] Example 3

[0070] A method for preparing a decellularized dermal matrix, the method comprising the following steps:

[0071] 1. Pretreatment of giant nevus tissue: The giant nevus tissue was placed in a cold chain preservation solution (composed of physiological saline and penicillin-streptomycin mixture, the stock solution of the mixture was a 100× concentrate, diluted 1× before use, with a final concentration of 100 U / mL penicillin + 100 μg / mL streptomycin; the mass concentration of antibiotics in the cold chain preservation solution was 0.7%). Under aseptic conditions, the epidermis and fat layer were removed (operation as in Example 1), and a 1 mm thick dermis was cut as experimental material.

[0072] 2. Decellularization: The pretreated giant nevus tissue was subjected to repeated freeze-thaw cycles (frozen at -90℃ for 5 h, thawed at 35℃ for 2 h, for a total of 3 times); then placed in sterile isotonic saline eluent and ultrasonically oscillated (frequency 30 kHz, eluent changed every 7 h, continuous oscillation and elution for 70 h) to obtain the decellularized giant nevus tissue.

[0073] 3. Depigmentation treatment: The decellularized giant nevus tissue was treated with 1% hydrogen peroxide solution at 35°C for 60 min, and then washed repeatedly with isotonic saline to obtain the depigmented giant nevus tissue.

[0074] 4. Stabilization and sterilization: The depigmented giant nevus tissue was uniformly irradiated with ultraviolet light at a wavelength of 365 nm for 30 seconds for stabilization treatment; then sterilized by ethylene oxide method to obtain decellularized dermal matrix.

[0075] Example 4

[0076] A method for preparing a decellularized dermal matrix, the method comprising the following steps:

[0077] 1. Pretreatment of giant nevus tissue: The giant nevus tissue was placed in a cold chain preservation solution (composed of physiological saline and penicillin-streptomycin mixture, the stock solution of the mixture was a 100× concentrate, diluted 1× before use, with a final concentration of 100 U / mL penicillin + 100 μg / mL streptomycin; the mass concentration of antibiotics in the cold chain preservation solution was 1.5%). Under aseptic conditions, the epidermis and fat layer were removed (operation as in Example 1), and a 1 mm thick dermis was cut as experimental material.

[0078] 2. Decellularization: The pretreated giant nevus tissue was subjected to repeated freeze-thaw cycles (frozen at -70℃ for 7 h, thawed at 40℃ for 0.5 h, for a total of 5 times); then placed in sterile isotonic saline eluent and ultrasonically oscillated (frequency 50 kHz, eluent changed every 5 h, continuous oscillation and elution for 75 h) to obtain the decellularized giant nevus tissue.

[0079] 3. Depigmentation treatment: The decellularized giant nevus tissue was treated with 3% hydrogen peroxide solution at 40°C for 20 min, and then washed repeatedly with isotonic saline to obtain the depigmented giant nevus tissue.

[0080] 4. Stabilization and sterilization: The depigmented giant nevus tissue was uniformly irradiated with ultraviolet light at a wavelength of 365 nm for 30 seconds for stabilization treatment; then sterilized by ethylene oxide method to obtain decellularized dermal matrix.

[0081] Comparative Example

[0082] Comparative Example 1

[0083] The decellularized dermal matrix was prepared in a manner similar to that in Example 1, with the only difference being:

[0084] The decellularization process was replaced by immersing the giant nevus tissue in a 5% sodium chloride solution at a material-to-liquid mass-to-volume ratio of 1 g: 12 mL, decellularizing for 12 h under stirring at 150 rpm, and then washing three times with water for injection.

[0085] Comparative Example 2

[0086] The decellularized dermal matrix was prepared in a manner similar to that in Example 1, except that no stabilization treatment was performed.

[0087] Comparative Example 3

[0088] The decellularized dermal matrix was prepared in a manner similar to that in Example 1, except that ultrasonic vibration was not performed.

[0089] Experimental Example

[0090] Experimental Example 1: SEM and HE staining tests

[0091] Scanning electron microscopy (SEM) and hematoxylin and eosin (HE) staining were performed on the untreated giant nevus tissue, as well as the depigmented giant nevus tissues from Examples 1 and 2. The test results are as follows: Figure 3 As shown.

[0092] Figure 3 In the images: A is a scanning electron microscope image of untreated ADM (giant nevus tissue), showing the presence of melanin granules within the tissue; B is an HE-stained image (40×) of untreated ADM, showing intact collagen fibers, indicating that the decellularization process of this invention does not damage the collagen fibers in the giant nevus tissue; C is a nevus cell stained image (40×) of untreated ADM, showing no residual nevus cells.

[0093] D is a scanning electron microscope image of MFADM from Example 1 (H2O2 depigmentation). No melanin granules remain in the tissue, indicating that the H2O2 depigmentation treatment of the present invention can effectively remove melanin from the giant nevus tissue. E is an HE staining image (40×) of MFADM from Example 1. Combined with image D, it can be seen that the collagen fibers in the tissue are intact, the three-dimensional network structure is intact, and the pores are evenly distributed, indicating that the H2O2 depigmentation treatment does not damage the collagen fibers and can retain the three-dimensional structure that is conducive to cell attachment and migration, which is suitable for subsequent transplantation needs. F is a staining image (40×) of nevus cells from MFADM from Example 1. No nevus cells are observed, indicating that the H2O2 depigmentation treatment can significantly remove nevus cells.

[0094] G is a scanning electron microscope image of MFADM from Example 2 (NaClO depigmentation), showing no melanin granules in the tissue; H is a HE-stained image (40×) of MFADM from Example 2, showing intact collagen fibers; I is a staining image (40×) of nevus cells from MFADM from Example 2, showing no residual nevus cells. These results confirm that the NaClO depigmentation treatment of this invention can effectively remove melanin and nevus cells without damaging the integrity of collagen fibers.

[0095] Experiment Example 2: Biocompatibility Test

[0096] The effects of the acellular dermal matrix MFADM (prepared in Examples 1 and 2) on the proliferation and adhesion of fibroblasts were evaluated using CCK-8 proliferation assays, live / dead cell staining, and cell adhesion rate assays. The test results are as follows: Figure 4 As shown.

[0097] Figure 4 In the middle: A to D are live / dead cell staining photographs (10×), which are, in order, the blank control group, Example 1 (H2O2 depigmented MFADM) group, the allogeneic ADM group, and Example 2 (NaClO depigmented MFADM) group; E to H are live / dead cell staining photographs (4×), which are, in order, the blank control group, Example 1 group, the allogeneic ADM group, and Example 2 group.

[0098] As shown in A to D, each ADM sample exhibits the growth of live cells labeled with green fluorescence, and the green fluorescence intensity of the live cells in Example 1 group is higher than that in Example 2 group. As shown in E to H, fibroblasts can penetrate into the ADM, and the number of fibroblasts in Example 1 group is greater than that in Example 2 group. The above results indicate that the MFADM prepared in Examples 1 and 2 has no obvious cytotoxicity, exhibits active cell proliferation and good adhesion ability, confirming that the decellularized dermal matrix prepared by the method of this invention has excellent biocompatibility. Furthermore, the MFADM prepared by H2O2 depigmentation treatment is more conducive to the growth and proliferation of live cells and fibroblasts than the sample treated with NaClO depigmentation.

[0099] Experiment Example 3: Biomechanical Testing

[0100] Biomechanical tests were performed on normal skin tissue and the decellularized dermal matrix prepared in Examples 1 and 2, respectively. Compression, tension, and tension cycle tests were used to evaluate the physical properties of the decellularized dermal matrix. The test results are as follows: Figure 5 As shown.

[0101] Figure 5 In the figures: A represents the compression test result, B represents the tensile test result, C represents the tensile cycle test result of Example 2 (NaClO depigmented MFADM), and D represents the tensile cycle test result of Example 1 (H2O2 depigmented MFADM).

[0102] Depend on Figure 5 It can be seen that, compared with the NaClO depigmentation group, the tensile strength and compressive strength of the H2O2 depigmentation MFADM group are closer to those of the control group (normal skin tissue), indicating that H2O2 depigmentation treatment is more conducive to preserving the biomechanical properties of giant nevus tissue.

[0103] Experiment Example 4: Decolorization effect, cytotoxicity and mechanical property testing

[0104] The decellularized dermal matrices prepared in Examples 1-4 and Comparative Examples 1-3 were tested for depigmentation effect, in vitro cytotoxicity / survival rate, and mechanical properties. In vitro cytotoxicity / survival rate was tested according to GB / T16886.5-2017 "Biological Evaluation of Medical Devices: Part 5: In Vitro Cytotoxicity Tests". Cell viability was quantitatively detected using 100% sample test solution, and the test results are shown in Table 1.

[0105] Table 1

[0106]

[0107] As shown in Table 1, the decellularized dermal matrices prepared in Examples 1-4 all achieved complete decolorization, while Comparative Examples 1 and 3 only achieved basic decolorization, and Comparative Example 2 achieved complete decolorization. Comparing the decolorization effects of Examples 1-4 with Comparative Example 1, it is evident that the synergistic scheme of "decellularization treatment + depigmentation treatment" adopted in this invention can significantly improve the decolorization effect. Comparing Examples 1-4 with Comparative Example 3, it is evident that the combined operation of "repeated freeze-thaw cycles + ultrasonic vibration" in the decellularization treatment is the key to ensuring excellent decolorization results.

[0108] In terms of mechanical properties, the tensile and compressive strengths of Examples 3 and 4 are similar to those of Example 1, indicating that the biomechanical properties of the decellularized dermal matrix prepared in these two examples meet the requirements for medical dermal matrix. The tensile strength (2.0 MPa) of Comparative Example 2 is much lower than that of Example 1 (2.9 MPa), and the compressive strength (3.5 MPa) is much higher than that of Example 1 (1.5 MPa), which cannot meet the medical requirements. This confirms that the stabilization treatment can effectively preserve the biomechanical properties of giant nevus tissue and ensure its medical suitability. The tensile strength of Comparative Example 3 is lower than that of Example 1, and the compressive strength is higher than that of Example 1, further proving that the synergistic effect of "repeated freeze-thaw + ultrasonic vibration" in the decellularization treatment is a necessary condition for preserving excellent biomechanical properties.

[0109] The present invention has been described in detail above with reference to specific embodiments and exemplary examples; however, these descriptions should not be construed as limiting the present invention. Those skilled in the art will understand that various equivalent substitutions, modifications, or improvements can be made to the technical solutions and embodiments of the present invention without departing from the spirit and scope of the invention, and all such modifications and improvements fall within the scope of the present invention. The scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for preparing acellular dermal matrix, characterized in that, The preparation method includes the following steps: Step 1: Place the giant nevus tissue in a cold chain preservation solution and remove the epidermis and fat layer of the giant nevus tissue under sterile conditions to obtain the pretreated giant nevus tissue. Step 2: Repeatedly freeze and thaw the pretreated giant nevus tissue, and then treat it with ultrasonic vibration to obtain decellularized giant nevus tissue; Step 3: Depigment the decellularized giant nevus tissue to obtain depigmented giant nevus tissue; Step 4: Stabilize the depigmented giant nevus tissue and then sterilize it to obtain decellularized dermal matrix.

2. The preparation method according to claim 1, characterized in that, In step 1, The cold chain preservation solution includes physiological saline and antibiotics, wherein the antibiotics are a penicillin-streptomycin mixed solution; The antibiotic has a mass concentration of 0.7-1.5% in the cold chain preservation solution.

3. The preparation method according to claim 2, characterized in that, In step 2, The conditions for repeated freeze-thaw cycles are as follows: freeze for 5 to 7 hours at a temperature of -90 to -70°C, followed by thawing for 0.5 to 2 hours at a temperature of 35 to 40°C. Each freeze-thaw cycle includes one freeze-thaw and one thawing, and the number of freeze-thaw cycles is 3 to 5.

4. The preparation method according to claim 1, characterized in that, In step 2, The conditions for ultrasonic oscillation are as follows: the giant nevus tissue after repeated freeze-thaw cycles is placed in sterile isotonic saline elution solution for ultrasonic oscillation at a frequency of 30-50 kHz. The sterile isotonic saline elution solution is replaced every 5-7 hours, and the oscillation is performed continuously for 70-75 hours.

5. The preparation method according to claim 1, characterized in that, In step 3, The decolorization treatment includes hydrogen peroxide decolorization treatment or acidic bleaching agent decolorization treatment; The conditions for the hydrogen peroxide depigmentation treatment are as follows: using hydrogen peroxide with a mass concentration of 1-3%, treating at a temperature of 35-40°C for 20-60 minutes, followed by multiple washes with isotonic physiological saline.

6. The preparation method according to claim 5, characterized in that, In step 3, The conditions for the acidic bleaching depigmentation treatment are as follows: use sodium hypochlorite with a mass concentration of 1-3% and treat at a temperature of 35-40°C for 20-60 minutes, followed by multiple washes with isotonic physiological saline.

7. The preparation method according to claim 1, characterized in that, In step 4, The stabilization treatment conditions are as follows: the depigmented giant nevus tissue is uniformly irradiated with ultraviolet light with a wavelength of 365nm for 20–40 seconds.

8. A decellularized dermal matrix prepared by the method according to any one of claims 1 to 7.

Images